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- PDB-5muu: dsRNA bacteriophage phi6 nucleocapsid -

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Basic information

Entry
Database: PDB / ID: 5muu
TitledsRNA bacteriophage phi6 nucleocapsid
Components
  • Major inner protein P1
  • Major outer capsid protein
  • Packaging enzyme P4
KeywordsVIRUS / icosahedral virus capsid shell
Function / homology
Function and homology information


T=13 icosahedral viral capsid / T=2 icosahedral viral capsid / viral procapsid / viral genome packaging / viral inner capsid / viral outer capsid / virion component => GO:0044423 / ribonucleoside triphosphate phosphatase activity / viral capsid / nucleoside-triphosphate phosphatase ...T=13 icosahedral viral capsid / T=2 icosahedral viral capsid / viral procapsid / viral genome packaging / viral inner capsid / viral outer capsid / virion component => GO:0044423 / ribonucleoside triphosphate phosphatase activity / viral capsid / nucleoside-triphosphate phosphatase / viral nucleocapsid / RNA binding / ATP binding / identical protein binding
Similarity search - Function
: / Major inner capsid protein P1 / Packaging enzyme P4 / ATPase P4 of dsRNA bacteriophage phi-12 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Major outer capsid protein / Packaging enzyme P4 / Major inner protein P1
Similarity search - Component
Biological speciesPseudomonas phage phi6 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsSun, Z. / El Omari, K. / Sun, X. / Ilca, S.L. / Kotecha, A. / Stuart, D.I. / Poranen, M.M. / Huiskonen, J.T.
CitationJournal: Nat Commun / Year: 2017
Title: Double-stranded RNA virus outer shell assembly by bona fide domain-swapping.
Authors: Zhaoyang Sun / Kamel El Omari / Xiaoyu Sun / Serban L Ilca / Abhay Kotecha / David I Stuart / Minna M Poranen / Juha T Huiskonen /
Abstract: Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by ...Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by calcium ions but its biomechanics remain poorly understood. Here, we describe the near-atomic resolution structure of the φ6 double-shelled particle. The outer T=13 shell protein P8 consists of two alpha-helical domains joined by a linker, which allows the trimer to adopt either a closed or an open conformation. The trimers in an open conformation swap domains with each other. Our observations allow us to propose a mechanistic model for calcium concentration regulated outer shell assembly. Furthermore, the structure provides a prime exemplar of bona fide domain-swapping. This leads us to extend the theory of domain-swapping from the level of monomeric subunits and multimers to closed spherical shells, and to hypothesize a mechanism by which closed protein shells may arise in evolution.
History
DepositionJan 14, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 22, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Experimental preparation / Category: em_sample_support / em_software
Item: _em_sample_support.grid_type / _em_software.image_processing_id ..._em_sample_support.grid_type / _em_software.image_processing_id / _em_software.imaging_id / _em_software.name
Revision 1.2Feb 20, 2019Group: Advisory / Data collection / Derived calculations
Category: em_admin / pdbx_data_processing_status ...em_admin / pdbx_data_processing_status / pdbx_database_proc / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _em_admin.last_update
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
  • Imaged by Jmol
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  • Biological unit as icosahedral 23 hexamer
  • Imaged by Jmol
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-3571
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  • Superimposition on EM map
  • EMDB-3571
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerMolecule:
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Assembly

Deposited unit
A: Major inner protein P1
B: Major inner protein P1
C: Packaging enzyme P4
D: Major outer capsid protein
E: Major outer capsid protein
F: Major outer capsid protein
G: Major outer capsid protein
H: Major outer capsid protein
I: Major outer capsid protein
J: Major outer capsid protein
K: Major outer capsid protein
L: Major outer capsid protein
M: Major outer capsid protein


Theoretical massNumber of molelcules
Total (without water)365,54413
Polymers365,54413
Non-polymers00
Water0
1
A: Major inner protein P1
B: Major inner protein P1
C: Packaging enzyme P4
D: Major outer capsid protein
E: Major outer capsid protein
F: Major outer capsid protein
G: Major outer capsid protein
H: Major outer capsid protein
I: Major outer capsid protein
J: Major outer capsid protein
K: Major outer capsid protein
L: Major outer capsid protein
M: Major outer capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)21,932,642780
Polymers21,932,642780
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
Buried area34980 Å2
ΔGint-186 kcal/mol
Surface area154630 Å2
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Major inner protein P1
B: Major inner protein P1
C: Packaging enzyme P4
D: Major outer capsid protein
E: Major outer capsid protein
F: Major outer capsid protein
G: Major outer capsid protein
H: Major outer capsid protein
I: Major outer capsid protein
J: Major outer capsid protein
K: Major outer capsid protein
L: Major outer capsid protein
M: Major outer capsid protein
x 5


  • icosahedral pentamer
  • 1.83 MDa, 65 polymers
Theoretical massNumber of molelcules
Total (without water)1,827,72065
Polymers1,827,72065
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Major inner protein P1
B: Major inner protein P1
C: Packaging enzyme P4
D: Major outer capsid protein
E: Major outer capsid protein
F: Major outer capsid protein
G: Major outer capsid protein
H: Major outer capsid protein
I: Major outer capsid protein
J: Major outer capsid protein
K: Major outer capsid protein
L: Major outer capsid protein
M: Major outer capsid protein
x 6


  • icosahedral 23 hexamer
  • 2.19 MDa, 78 polymers
Theoretical massNumber of molelcules
Total (without water)2,193,26478
Polymers2,193,26478
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Major inner protein P1


Mass: 85080.711 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Pseudomonas phage phi6 (bacteriophage) / References: UniProt: P11126
#2: Protein Packaging enzyme P4


Mass: 35198.426 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Pseudomonas phage phi6 (bacteriophage)
References: UniProt: P11125, nucleoside-triphosphate phosphatase
#3: Protein
Major outer capsid protein / Protein P8


Mass: 16018.418 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) Pseudomonas phage phi6 (bacteriophage) / References: UniProt: P07579

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
Crystal symmetry∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name H-M: P1

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Sample preparation

ComponentName: Pseudomonas phage phi6Pseudomonas virus phi6 / Type: VIRUS
Details: The viral envelope was removed by Triton X-114 extraction
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Details of virusEmpty: NO / Enveloped: YES / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: Pseudomonas syringae / Strain: pv.phaseolicola HB10Y
Virus shell
IDEntity assembly-IDNameDiameter (nm)Triangulation number (T number)
11Outer shell56513
21Inner shell5001
Buffer solutionpH: 7.2
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 160000 X / Calibrated magnification: 37037 X / Nominal defocus max: 300 nm / Calibrated defocus max: 3000 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 120 K / Temperature (min): 80 K
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 0.73 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 900
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 3710 / Height: 3710 / Movie frames/image: 22 / Used frames/image: 1-22

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Processing

SoftwareName: PHENIX / Version: dev_2044: / Classification: refinement
EM software
IDNameVersionCategoryDetailsFitting-ID
1ETHAN1.2particle selection
2SerialEMimage acquisition
4CTFFIND3CTF correctionCTFFIND was used to determine CTF parameters
5RELION1.3CTF correctionRELION was used to apply CTF correction
8Coot0.8.7model fitting1
10RELION1.3initial Euler assignment
11RELION1.3final Euler assignment
12RELION1.3classification
13RELION1.33D reconstruction
21Coot0.8.7model refinement1
22PHENIX1.10.1_2155model refinement1
42Coot0.8.7model fitting2
43Coot0.8.7model refinement2
44PHENIX1.10.1_2155model refinement2
64Coot0.8.7model fitting3
65Coot0.8.7model refinement3
66PHENIX1.10.1_2155model refinement3
Crystal symmetry∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name H-M: P1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 16466
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13291 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALThe structure of P1 was fitted in the map using COOT as a rigid body in two different positions corresponding to subunits P1A and P1B. P1A and P1B main-chains and side-chains were adjusted using manual and real space fitting in COOT. Structure was refined in Phenix.real_space_refine applying secondary structure, rotamer, and Ramachandran plot restraints.
2AB INITIO MODELREALThe structure of P8 (L3 to Y147) was built manually in COOT and side-chains were adjusted using manual and real space fitting in COOT. Structure was refined in Phenix.real_space_refine applying secondary structure, rotamer, and Ramachandran plot restraints.
3AB INITIO MODELThe structure of P4 C-terminus (R292 to L332) was built manually in COOT and side-chains were adjusted using manual and real space fitting in COOT. Structure was refined in Phenix.real_space_refine applying secondary structure, rotamer, and Ramachandran plot restraints.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
14K7H

4k7h

A11-760
22
33
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00923680
ELECTRON MICROSCOPYf_angle_d1.00332128
ELECTRON MICROSCOPYf_dihedral_angle_d7.53314416
ELECTRON MICROSCOPYf_chiral_restr0.053724
ELECTRON MICROSCOPYf_plane_restr0.0044173

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